Recent prospective clinical trials have underscored our lack of knowledge with regard to the effect of gender and sex hormones on the coronary vasculature, especially at the cellular/molecular level. Sex hormones exert profound influence on vascular smooth muscle physiology, including proliferation, ion channel activity and channel expression. Coronary smooth muscle (CSM) ion channel activity is central to both regulation of coronary blood flow and progression of vascular disease. We have demonstrated that CSM calcium and potassium channel activities are strongly influenced by gender. This study will determine the mechanism for gender-specific differences in CSM voltage-gated calcium (VGCC) and K channel activity and the consequent effects on intracellular calcium (Cai) regulation and vasoreactivity. The overall hypothesis is that coronary arterial reactivity in males is greater due to a testosterone (TST)-dependent increase in VGCC synthesis and PKC-dependent activity resulting in an enhanced propensity for coronary vasospasm (CVS). Both in vivo (intact, gonadectomized and hormone-replaced swine) and in vitro techniques will be used to determine the role of gender, TST and estrogen (E2) in regulating CSM ion channel activity and coronary arterial reactivity. Aim 1 will determine the mechanism of gender and sex hormone induced changes in VGCC activity and expression using electrophysiology, immunoblot and RT-PCR. As both vasoreactivity and VGCC activity are modulated by PKC, gender and sex hormone effects on PKC regulation of VGCC will also be examined. As the effect of VGCC activity on Cai and contraction is modulated via coupling to K channels and cellular calcium buffering, Aim 2 will determine the effect of sex-specific differences in VGCC activity on Ca-activated (BK) and voltage-dependent K current (Kv) and Cai using simultaneous voltage clamp and microfluorometry. The effect of sex-specific differences in VGCC activity on macro- and microvascular reactivity under both physiological and pathophysiological conditions will be assessed in vitro and in vivo in Aim 3. A model of CVS will be used to assess the role of sex-specific differences in VGCC activity on the progression and severity of vascular disease. The goal of this research is to determine gender-related cellular and molecular differences in CSM as they relate to gender differences in propensity for coronary disease.